Project Summary Clostridium difficile (Cd), a Gram-positive anaerobe, is a leading cause of both hospital- and community- acquired antibiotic associated diarrhea. Cd infection (CDI) typically occurs after treatment with broad-spectrum antibiotics disrupts the normal gut microbiota, allowing for Cd colonization. While CDI is a toxin-mediated disease, its capacity for transmission and, therefore, disease is dependent on spore formation and germination. Cd spores become metabolically active (germinate) when receptors packaged within the spore recognize host-specific molecules (e.g., bile salts, glycine) known as germinants. Currently, the mechanism of Cd germination is not completely understood. Many of the proteins required for germination in other spore forming bacteria, including known germinant receptors, are not encoded in the genomes of sequenced Cd strains. The receptor for the bile salt taurocholate (Tc), CspC, has been identified. However, the receptor(s) for the required co-germinant, glycine, remains unknown. There is a fundamental gap in our understanding of the mechanism controlling the requirement of glycine for efficient germination of Cd spores. Addressing this gap has the potential for the development of therapeutics that can improve the outcome of CDI and could provide new targets for disruption of persistent infections, reducing the burden of recurrent CDI. The central hypothesis is that glycine is sensed in the environment through specific interactions with receptors within the spore. These interactions trigger core rehydration and subsequent release of calcium-dipicolinic acid (Ca-DPA). In turn, Ca-DPA transduces the germination signal from the spore core inducing cortex hydrolysis by mature SleC. Guided by substantial preliminary data, this hypothesis will be tested in the following two specific aims: 1) Define the role of proteins essential for Tc-Glycine induced germination. The applicant has identified a protein that is highly expressed during sporulation that is essential for glycine-induced germination, termed GsgA. The goal of this aim is to define the role of GsgA in Cd germination and pathogenesis in a murine model. 2) Characterization of the mechanism of Tc-CaDPA induced germination. The applicant has discovered a novel method to induce efficient germination in Cd. By using Tc and CaDPA the applicant is able to circumvent the need for glycine. The goal of this aim is to elucidate the mechanism for Tc-CaDPA induced germination. The proposed research is significant because it will provide insight into the unknown mechanisms of Cd germination. Ultimately, this knowledge will provide an important foundation for the use of novel therapeutic targets that may improve outcomes for patients with recurrent CDI.